In molecular biology, increasing use is being made of biochips with which discoveries about organisms and tissue can be made in a rapid fashion. The detection of (bio)chemical reactions, that is to say the detection of biologically relevant molecules in a defined study material, is extremely important for the biosciences and medical diagnosis. In this scope, the development of so-called biochips is being constantly pursued. Such biochips are usually miniaturized hybrid functional elements with biological and technical components, in particular biomolecules which are immobilized on a surface of a biochip base module and are used as specific interaction partners. The structure of these functional elements often has rows and columns. The term “microarrays” is then used. Since thousands of biological or biochemical functional elements can be arranged on one chip, they generally need to be fabricated using microtechnological methods.
Particularly suitable as biological and biochemical functional elements are: DNA, RNA, PNA, (in the case of nucleic acids and chemical derivatives thereof, there may for example be single strands, triplex structures or combination thereof present), saccharides, peptides, proteins (for example antibodies, antigens, receptors), derivatives from combinatorial chemistry (for example organic molecules), cell components (for example organelles), cells, multicellular organisms, or cell groups.
So-called microarrays are the most widespread variant of biochips. They are small wafers (“chips”) for example of glass, gold, plastic or silicon. In order to detect corresponding biological or biochemical (binding) reactions, for example, small amounts of various solubilized capture molecules, for example a known nucleic acid sequence, are fixed on the surface of the biochip base module in the form of very small droplets, so-called dots, in a point-like and matrix-like fashion.
In practice, a few hundred to a few thousand droplets are used per chip. An analyte to be studied, which may for example contain fluorescence-labelled target molecules, is then pumped over this surface. This generally leads to various chemical (binding) reactions between the target molecules contained in the analyte and the fixed or immobilized capture molecules. As mentioned above, the target molecules are labelled with dyestuff molecule components, usually fluorochromes, in order to observe these reactions or bindings. The presence and the intensity of light which is emitted by the fluorochromes provides information about the progress of the reaction or binding in the individual droplets on the substrate, so that conclusions can be drawn about the presence and/or the property of the target molecules and/or capture molecules. When the corresponding fluorescence-labelled target molecules of the analyte react with or bind to the capture molecules immobilized on the surface of the support substrate, this reaction or binding can be detected by optical excitation with a laser and measurement of the corresponding fluorescence signal.
Substrates with a high but defined porosity have many advantages over planar substrates as a basis for such biochips.
More detection reactions can take place on the greatly enlarged surface area. This increases the detection sensitivity for biological assays. When the target molecules dissolved in the analyte are pumped through the channels between the front and back sides of the porous substrate, they are brought in close spatial contact with the surface of the substrate (<10 μm). On this size scale, diffusion is a very effective transport process which quickly covers the distance between a target molecule to be detected and the capture molecules immobilized on the surface. The rate of the binding reaction can thereby be increased so that the duration of the detection method can be significantly shortened.
Electrochemically produced porous silicon is an example of a substrate with such a defined porosity (cf. DE 42 02 454 A1, EP 0 553 465 A1 or DE 198 20 756 A1).
Many of the analytical methods currently used in active-agent research and clinical diagnosis employ optical methods for the detection of binding events between a substance to be detected and capture molecules (for example DNA hybridizations, antigen-antigen interactions and protein interactions). The substance to be detected is in this case provided with a marker which fluoresces after excitation with light of a suitable wavelength (fluorescence method) or which initiates a chemical reaction that in turn produces light (chemiluminescence method).
When the substance to be detected, that is to say the target molecule, binds with the immobilized capture molecule on the surface, then this can be detected optically, for example by means of luminescence. The term “luminescence,” is in this case intended to mean the spontaneous emission of photons in the ultraviolet to infrared spectral range. The luminescent excitation mechanisms may be optical or non-optical in nature, for example electrical, chemical, biochemical and/or thermal excitation processes. Therefore, in particular, chemi-, bio- and electro-luminescence as well as fluorescence and phosphorescence are intended to be covered by the term “luminescence,” in the scope of this invention.
Porous substrates with a high optical density and low reflectivity, for example porous silicon whose reflectivity is 50 to 70% in the visible range of the spectrum, however, do not give the expected results in conjunction with fluorescence or chemiluminescence methods in so far as the experimentally observed light-signal yield falls far short of the theoretically achievable values. The reasons for reduced experimentally determined light-signal yields compared with the theoretical values when such porous substrates are used are, on the one hand, problems with emitting the fluorescence of the substance or binding to be studied, and on the other hand—when a fluorescence method is used—problems with the optically exciting the fluorescence.
If (luminescent) light is produced throughout the volume of the pores, then the reflectivity of the pore walls is a crucial factor with a view to effective delivery of the optical signal to the surface. In the case of chemiluminescence, the light signal is radiated isotropically in all directions of space. Consequently, only a very small proportion of the generated light radiates directly in the aperture angle of the individual pore. All other optical paths are reflected several times by the walls of the pores before they reach the opening of the pore in question. Even with reflectivities which are only a little less than 100%, however, the intensity of a signal will be greatly reduced after multiple reflections. This means that this proportion of the generated signal will be greatly attenuated on its way out of the pore, and can then scarcely make any contribution to the overall signal.
Attenuation due to multiple reflections by the pore walls, which has already been described in connection with the problems of exciting the fluorescence, furthermore constitutes a serious problem for emitting the luminescence. Only fluorophors (fluorescent substances in the analyte) which radiate directly towards the pore opening are available unattenuated for a fluorescence signal. All the other optical paths are reflected at least once by the walls of the pores before they reach the opening of the pore. Even with reflectivities which are only a little less than 100%, these multiple reflections will lead to a significant attenuation of the optical signal to be detected.
In order to resolve the aforementioned problems of intensity attenuation due to multiple reflections, it has been proposed to arrange reflection layers on the pore walls in order to reduce the reflection losses, so that the excitation and emission light can be delivered better from the pores. But this solution approach does not lead to any significant improvement of the signal yield.